Kinetics and Mechanism of the Citrate Synthase from the Thermophilic Archaeon Thermoplasma acidophilum

Abstract

The kinetics and mechanism of the citrate synthase from a moderate thermophile, Thermoplasma acidophilum (TpCS), are compared with those of the citrate synthase from a mesophile, pig heart (PCS). All discrete steps in the mechanistic sequence of PCS can be identified in TpCS. The catalytic strategies identified in PCS, destabilization of the oxaloacetate substrate carbonyl and stabilization of the reactive species, acetyl-CoA enolate, are present in TpCS. Conformational changes, which allow the enzyme to efficiently catalyze both condensation of acetyl-CoA thioester and subsequently hydrolysis of citryl-CoA thioester within the same active site, occur in both enzymes. However, significant differences exist between the two enzymes. PCS is a characteristically efficient enzyme: no internal step is clearly rate-limiting and the condensation step is readily reversible. TpCS is a less efficient catalyst. Over a broad temperature range, inadequate stabilization of the transition state for citryl-CoA hydrolysis renders this step nearly rate-limiting for the forward reaction of TpCS. Further, excessive stabilization of the citryl-CoA intermediate renders the condensation step nearly irreversible. Values of substrate and solvent deuterium isotope effects are consistent with the kinetic model. Near its temperature optimum (70 degrees C), there is a modest increase in the reversibility of the condensation step for TpCS, but reversibility still falls short of that shown by PCS at 37 degrees C. The root cause of the catalytic inefficiency of TpCS may lie in the lack of protein flexibility imposed by the requirement for thermal stability of the protein itself or its temperature-labile substrate, oxaloacetate.

Crystal Structures of Escherichia coli KDO8P Synthase Complexes Reveal the Source of Catalytic Irreversibility

“…In a recent study of citrate synthase (CS), protein flexibility during catalysis was found to be a major determinant in the rate of catalysis. 33 CS performs a Claisen condensation between oxaloacetate and actetyl-coenzyme A to form citrate and coenzyme A. When CS from a moderate thermophile was compared to a mammalian CS, it was found to be significantly more rigid, probably in order to cope with the higher temperature.…”

“…18,34,35 On the basis of the analysis of CS, one would expect that the extensive flexibility in KDO8PS would result in a significant rate of reversibility. 33 The E. coli KDO8PS avoids this problem by two methods. A major conformational change, the closing of L7, occurs only as result of PEP binding followed by A5P binding, in the correct orientation.…”

Role of the Bacillus methanolicus Citrate Synthase II Gene, citY , in Regulating the Secretion of Glutamate in l -Lysine-Secreting Mutants

“…The crystal structures of CS have been determined and compared for organisms capable of growth over a very wide range of temperatures, from the Antarctic bacterium Arthrobacter sp. strain DS2-3R (maximum growth at 30°C) (42), mesophilic organisms such as chicken and porcine heart (37°C), thermotolerant organisms such as Thermoplasma acidophilum (55°C) (28), and the extreme thermophiles Sulfolobus solfataricus (88°C) (8), and Pyrococcus furiosus (100°C) (28).…”

The Partial Substrate Dethiaacetyl-Coenzyme A Mimics All Critical Carbon Acid Reactions in the Condensation Half-Reaction Catalyzed by Thermoplasma acidophilum Citrate Synthase

“…Dethiaacetyl-CoA was prepared as described (21). The Tp CS-expressing pRec7-ArCS plasmid (3) was mutated with a QuikChange kit (Stratagene) and oligodeoxynucleotides (Supporting information, Table S1) from IDT to furnish plasmids pJK381 (D317G– Tp CS) and pJK382 (D317N– Tp CS). For experiments in D 2 O solutions, the pD was calculated from the pH meter reading (calibrated with standards in H 2 O) by adding 0.4 (22).…”